LINEs Mobilize SINEs in the Eel through a Shared 3′ Sequence Masaki Kajikawa, Norihiro Okada  Cell  Volume 111, Issue 3, Pages 433-444 (November 2002) DOI: 10.1016/S0092-8674(02)01041-3

Figure 1 Retrotransposition Assay for UnaL2 (A) Schematic representation of UnaL2 and UnaSINE1 characterized from the eel genome and an alignment of their 3′ conserved tail regions. The single ORF comprises the shaded boxes, and putative endonuclease (EN) and reverse transcriptase (RT) domains are indicated. The 5′ and 3′ untranslated regions (UTRs) are indicated by open boxes. The 3′ terminal repeats in UnaL2 and UnaSINE1 are underlined by a single line in the sequence alignment. The putative upper-stem regions are underlined by double lines. The putative poly A signal in the UnaL2 sequence is indicated by a dotted underline. (B) Schematic of the retrotransposition assay in HeLa cells using the mneol construct. PCMV, cytomegalovirus promoter. UnpA, putative UnaL2 poly A signal. SVpA, SV40 poly A signal. (C) Overview of the experimental procedure. Cell 2002 111, 433-444DOI: (10.1016/S0092-8674(02)01041-3)

Figure 2 Retrotransposition of UnaL2s Can Be Assayed by Using HeLa Cells (A) Results of retrotransposition of UnaL2s using four different plasmids, 1–29 (wild-type), RTm-60 (the RT gene mutation; D694Y), ENm-1 (the EN gene mutation; E73A), and 4–5 (a deletion of the 3′ tail of UnaL2). The sites of mutations of D694Y and E73A in UnaL2 are identical with those of D702Y and E43A in human L1 (Feng et al., 1996; Moran et al., 1996). N, the number of independent transfection experiments with each construct. RF, retrotransposition frequency calculated as described in Experimental Procedures. Images show each 100 mm plate with G-418R cell colonies selected from approximately 2∼4 × 106 HygR cells. (B) Southern hybridization of genomic DNAs from G-418R cells. Genomic DNAs from four independent clonal cell lines (b-e) were analyzed. Genomic DNA from intact HeLa cells was used as a negative control (a). (C) Schematic representation of structures of retrotransposed UnaL2 loci. HG, human genome sequence. PCMV, cytomegalovirus promoter. UnpA, putative UnaL2 poly A signal. SVpA, SV40 poly A signal. Int, Chr, and TSA represent the presence or absence of the intron, the chromosomal location of insertion site, and characteristics of target site alteration of the insertion site, respectively. The probe sequence used in Figure 2B is indicated by a thick bar. Cell 2002 111, 433-444DOI: (10.1016/S0092-8674(02)01041-3)

Figure 3 Mutations within the 3′ Conserved Tail Region Affect Retrotransposition Predicted secondary structure of the 3′ conserved tail region of UnaL2 RNA. Constructs with mutations used in retrotransposition assays and their retrotransposition frequencies are indicated. Retrotransposition assays were performed as described in Figures 1B and 1C. Retrotransposition frequencies (underlined) were calculated as described in Experimental Procedures. Cell 2002 111, 433-444DOI: (10.1016/S0092-8674(02)01041-3)

Figure 4 The 3′ Conserved Tail of UnaL2 Is Recognized by the UnaL2 Enzymatic Machinery in trans (A) Schematic of plasmids used to assay trans retrotransposition in HeLa cells. Constructs used as test sequences are represented in comparison to the UnaL2 sequence at the top. UnaL2 Middle, UnaL2 3′UTR, and UnaL2 Tail were derived from the middle region, the 3′UTR, and the 3′ tail of UnaL2, respectively. Diagram of the plasmids used for trans and cis retrotransposition are shown below the test sequence schematics. The test sequences were inserted into the first plasmid containing the retrotransposition reporter cassette (mneol), and the UnaL2 RT was expressed by the second plasmid (pUR8). Plasmids pUR8/RTm and pUR8/ENm are pUR8 variants that have the same missense mutation as plasmids RTm-60 and ENm-1, respectively. Plasmids RTm-60 and 1–29 are the same plasmids described in Figure 2A. The cis retrotransposition constructs served as positive controls and the retrotransposition frequency is shown for comparison. N, the number of independent transfection experiments for each construct. RF, retrotransposition frequency calculated as described in Experimental Procedures. Approximately 1∼4 × 106 HygR and HisR cells were seeded per 100 mm plate and G-418 selection was performed. In the case of UnaL2 Tail as a test sequence, the mean number of G-418R colonies per 100 mm plate was 114.5 ± 20.0 and the RF was calculated as 49.3 ± 12.7. When no G-418R colony was observed, the RF was calculated as < 1.0. (B) RT expression plasmids having inefficient cis retrotransposition frequencies increase retrotransposition frequencies in trans. The UnaL2 Tail segment was used in the mneol plasmid. The following constructs were used for the UnaL2 expression plasmid: pUR8 (wild-type), lm (loop mutant; the same mutant as loop-1 in Figure 3), sm (stem mutant; the same mutant as stem1-3 in Figure 3), and rd (repeat deletion mutant; the same mutant as no-rep in Figure 3). Cell 2002 111, 433-444DOI: (10.1016/S0092-8674(02)01041-3)

Figure 5 The 3′ Conserved Tail of UnaSINE1 Is Recognized by the UnaL2 Enzymatic Machinery in trans. (A) Schematic representation of the 3′ conserved region of three LINE/SINE partners. UnaL2 and UnaSINE1 from eel, Rsg-1 LINE and HpaI SINE from salmon (Ohshima et al., 1996), and CiLINE2 and Af1 SINE from cichlid fish (Terai et al., 1998). Sequences of the 3′ conserved region of UnaL2/UnaSINE1, Rsg-1 LINE/HpaI SINE, and CiLINE2/Af1 SINE are different from one another. (B) trans retrotransposition assay using the 3′ conserved region of LINE/SINE partners as the test sequences. The trans retrotransposition assay was performed as described in Figure 4A. The transcription of all test sequences is under the control of a CMV promoter and an SV40 poly A signal. N, the number of independent transfection experiments for each construct. RF, retrotransposition frequency calculated as described in Experimental Procedures. Approximately 2∼4 × 106 HygR and HisR cells were seeded per 100 mm plate and G-418 selection was performed. In the case of UnaSINE1 as a test sequence, the mean number of G-418R colonies per 100 mm plate was 84.5 ± 2.9 and the RF was calculated as 37.5 ± 2.5. When no G-418R colony was observed, the RF was calculated as < 0.5. The point mutation in box B of the SINE internal promoter (abolishes the pol III transcription of SINEs) is indicated by an asterisk. Images show each 100 mm plate with G-418R cell colonies selected from approximately 2∼4 × 106 HygR and HisR cells. (C) Schematic representation of structures of retrotransposed UnaSINE1 loci. Abbreviations are the same as those in the legend of Figure 2C. N.K., not known. Cell 2002 111, 433-444DOI: (10.1016/S0092-8674(02)01041-3)

Figure 6 Multiple 3′ Tail Repeats Are Required for Retrotransposition (A) Comparison of retrotransposition frequencies for UnaL2 constructs with varying numbers of [TGTAA] repeats. The retrotransposition assay was performed as described in Figures 1B and 1C. N, the number of independent transfection experiments for each construct. Rep. No., the number of 3′ [TGTAA] repeats. RF, retrotransposition frequency calculated as described in Experimental Procedures. Approximately 2 × 105 ∼4 × 106 HygR cells were seeded per 100 mm plate and G-418 selection was performed. In the case of rep3, the mean number of G-418R colonies per 100 mm plate was 77.0 ± 5.6 (2∼4 × 105 HygR cells per 100 mm plate) and the RF was calculated as 286.6 ± 74.5. (B) Many repeats can function during retrotransposition. The flanking UnaL2 sequence into which the repeats are inserted is shown. In the case of repran-3, the mean number of G-418R colonies per 100 mm plate was 4.5 ± 0.5 (2∼4 × 106 HygR cells per 100 mm plate) and the RF was calculated as 1.5 ± 0.4. In the case of repran-7, no G-418R colony was observed and the RF was < 0.5. In the case of no-rep, 0∼2 colonies per 100 mm plate were observed and the RF was < 0.6. Rep. Seq., the repeat sequence used to replace wild-type sequence. PWA, relative value of RFs for clones containing these repeats compared with the wild-type (rep3). Cell 2002 111, 433-444DOI: (10.1016/S0092-8674(02)01041-3)

Figure 7 The Sequence of the 3′ Terminal Repeat of Retrotransposed Copies of UnaL2s Is Altered During Retrotransposition (A and B) Compilation of the sequences of the 3′ terminal repeat and its flank of parental plasmids and retrotransposed copies of UnaL2s. Retrotransposition frequencies of repmut-1, 2, 3, and 4 are 119.1 ± 50.9, 555.1 ± 177.4, 67.1 ± 17.4, and 150.1 ± 30.6, respectively. Bold letters represent mutated nucleotides introduced into parental plasmids as they were observed in retrotransposed copies of UnaL2. The [TGTAA] units are underlined. The 3′ terminal repeats of UnaL2s in the parental plasmids are followed by a vector sequence (small letters), and the 3′ terminal repeats of retrotransposed copies are followed by various human genome sequences (boxed letters). Repeat units different from those of [TGTAA] are double underlined. (C) A template slippage model to explain the deviation of the 3′ tail repeat sequence from the parental sequence following retrotransposition. One of the retrotransposed copies from repmut-1 is used as an example. Vector, vector sequence. HG, human genome sequence. Step 1, transcription of UnaL2; Step 2, initiation of the reverse transcription using the first repeat as template and the 3′-OH of DNA as primer; Step 3, template slippage; Step 4, reinitiation of reverse transcription; Step 5, template slippage; Step 6, reinitiation of reverse transcription; Step 7, integration of the synthesized copy of UnaL2 DNA into the host genome after the completion of reverse transcription. Cell 2002 111, 433-444DOI: (10.1016/S0092-8674(02)01041-3)